Method and pharmaceutical composition for inhibiting pi3k/akt/mtor signaling pathway

ABSTRACT

The present invention relates to a pharmaceutical composition of PRCP antagonist, PREP antagonist, or PRCP-PREP dual antagonist. The present invention also relates to a pharmaceutical composition jointly using PRCP antagonist and mTOR antagonist, or jointly using PREP antagonist and mTOR antagonist, or jointly using PRCP-PREP dual antagonist and mTOR antagonist. In addition, the present invention also relates to a method for utilizing the pharmaceutical compositions to treat and prevent cancers and diseases related to insulin receptor substrate protein and PI3K/AKT/mTOR signal pathway.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage application of PCT/CN2013/001182filed Sep. 29, 2013 which claims the benefit of priority to Chinesepatent Application No. 20,210,379,418.9.

TECHNICAL FIELD

Drug combinations and methods for inhibiting insulin receptor substrateand PI3K/AKT/mTOR signaling pathways are provided. The present inventionrelates to inhibition of a PI3K (phosphatidylinositol kinase(Phosphoinositide 3-kinase)/AKT (protein kinase B)/mTOR (mammaliantarget of rapamycin) signaling pathway for treating or preventing adisease. More particularly, the invention relates PRCP(prolylcarboxypeptidase) antagonist (i.e. anti-PRCP agent), or PREP(prolyl endopeptidase) antagonist anti-PREP agent), or a pharmaceuticalcomposition of PREP/PRCP dual antagonist, a pharmaceutical compositioncomprising PREP antagonist or PRCP antagonist drugs and mTORantagonists, or a pharmaceutical composition comprising PRCP and PREPdual antagonists and mTOR antagonist. The present invention also relatesto the aforementioned pharmaceutical composition for treating orpreventing diseases associated with activated PI3K/AKT/mTOR signalingpathway. Furthermore, the present invention also relates to themolecular mechanisms of stability and degradation of the insulinreceptor substrate proteins and the use of PRCP antagonists, PREPantagonists, dual antagonists of PREP and PRCP for degradation ofinsulin receptor substrate proteins for treatment.

BACKGROUND

PRCP and PREP belong to prolyl peptidase family. Phylogenetic analysisshows that PRCP and PREP contain highly similar amino acid sequences,and have a similar enzyme function. They can cleave proline-containingsubstrates such as neuropeptide angiotensin II/III (Angll/III) andα-melanocyte stimulating hormone (α-MSH). PREP additionally cleavesnerve vasopressin (neurotensin) and gastrin-releasing hormone and otherneuropeptides. These neuropeptides can activate G protein-coupledreceptors (GPCR) and regulate the function of the receptor tyrosinekinase signaling pathway through the G protein-coupled receptors(Garcia-Horsman et al, (2007) Neutopeptides 41: 1-24; Rosenblum J S etal “(2003) Current Opinion in Chemical Biology, 7:496-504; Skidgel etal, (1998) Immunological Reviews, 161: 129-41. Rozengurt E et al, (2010)Clin Cancer Res; 16: 2505-11). Current research indicates that PRCPplays a role in obesity (Shariat-Madar B et al, (2010) Diabetes MetabSyndr Obes, 3: 67-78). Our previous study teaches that PRCP regulatesbreast cancer cell proliferation, autophagy, and resistance to the drugtamoxifen (Duan L et al, (2011) JBC, 286:2864-2876). PREP is alsoassociated with amnesia, depression and Alzheimer's disease (Rosenblum JS et al, (2003) Current Opinion in Chemical Biology 7:496-504). Cellgrowth and proliferation are regulated by a number of different factors,including the availability of nutrients, growth factors (such as insulinand insulin-like growth factor, etc.) as well as the availability of theenergy state of the cell, etc. PI3K/AKT/mTOR provide signal pathwayintegration of these factors to control cell growth and proliferation(Manning et al, (2007) Cell 129: 1261-1274; Engelman et al, (2006) NatRev Genet 7:606-619). Aberrant activation of PI3K/AKT/mTOR signalingpathway is considered to be the most common feature of all cancers(Engelman, J A, (2009) Nature Reviews/Cancer 9:550-562).

PI3K/AKT/mTOR signaling pathway is activated by RTKs (receptor tyrosinekinases), including the insulin receptor (IR), insulin-like growthfactor receptor (IGF-1R), platelet-derived growth factor receptor(PDGFR) and epidermal growth factor receptor (EGFR).

RTKs can activate PI3K directly or indirectly through insulin receptorsubstrate (IRS) that interacts with PI3K ρ85 subunit and furtheractivates PI3K p110 catalytic subunits (Markman et al., (2009) AnnOncol. 21 (4): 683-91).

P13K is an intracellular phosphatidylinositol kinase. There are threetypes of PI3K. Class I PI3Ks are mostly cytosolic, are heterodimerscomprised of a p110 catalytic subunit and an adaptor/regulatory subunit,and are further divided into two subclasses: Class IA PI3Ks consist of ap110 catalytic subunit that associates with an SH2 domain-containingsubunit p85, and is activated by the majority of tyrosine kinase-coupledtransmembrane receptors; class IB PI3K consists of a p101 regulatorysubunit that associates with p110γ catalytic subunit, and is activatedby heterotrimeric GPCR. (Katso et al. (2001) Annu. Rev. Cell Dev. Biol.17:615). Class II PI3Ks consist of three isoforms, as discussed herein.Class III PI3Ks utilize only phosphatidylinositol as a substrate, andplay an essential role in protein trafficking through the lysosome.(Volinia, et al. (1995) EMBO J. 14:3339).

Class IA PI3K activity is suppressed in cytosol by p85 regulatorysubunits that form heterodimers with the p110 catalytic subunit. IRSproteins (including IRS-1, IRS-1, IRS-3, IRS-4) are insulin receptor(IR) and insulin-like growth factor receptor (IGF-1R) adapter proteins.IR/IGF1R activates PI3K by regulating IRS protein tyrosinephosphorylation and subsequent interaction with PI3K p85 subunit. Manycancer tissues overexpress insulin receptor substrate IRS-1, whiletransgenic overexpression of IRS-1 or IRS-2 in mice caused breast cancertumorigenesis and metastasis (Metz, et al, (2011) Clin Cancer Res 17:206-211; Bergmann et al, (1996) Biochem Biophys Res Commun 220: 886-890;Dearth et al, (2006) Mol Cell Biol 26: 9302-9314). Tyrosinephosphorylation of IRS proteins is regulated by IR/IGF-1R and other RTKssuch as EGFR and ErbB3 which activate IRS proteins. IRS proteins arealso regulated by a number of serine/threonine kinases (for example.PKC, mTOR, S6K and ERK) that phosphorylate IRS proteins on serineleading to protein degradation and inhibition of IRS proteins (Copps etal (2012). Diabetologia. 55(10): 2565-2582). Degradation of insulinreceptor substrates by certain drugs results in cell death in melanoma(Reuveni et al (2013) Cancer Res 73: 4383-4394). IRS proteinsphosphorylated on tyrosine interact with the SH2 domain of p85 subunitresulting in recruitment of PI3K to membrane and release of theinhibitory effect of p85 leading to activation of PI3K. PI3Ks areenzymes that phosphorylate the 3-hydroxyl position of the inositol ringof phosphoinositides (“PIs”). Activated PI3K generatesphosphatidylinositol 3-phosphate (PI3P) that serves as a secondarymessenger in growth signaling pathways, influencing cellular eventsincluding cell survival, migration, motility, and proliferation;oncogenic transformation; tissue neovascularization; and intracellularprotein trafficking. PI3P activates the PI3K-dependent protein kinase-1(PDK1), which in turn activates the kinase AKT. AKT phosphorylatesdownstream target molecules to promote cell proliferation, survival andneovascularization. (Cantley et al. (1999) PNAS 96:4240) mTOR is animportant signaling molecule downstream of the PI3K/AKT pathway(Grunwald et al. (2002) Cancer Res. 62: 6141; Stolovich et al. (2002)Mol Cell Biol. 22: 8101). AKT-mediated phosphorylation inhibits the GAPactivity of TSC1/TSC2 toward the Rheb GTPase, leading to Rhebactivation. Rheb binds directly to mTOR, a process that is regulated byamino acids. Both amino acids and Rheb activation are required for mTORactivation. mTOR downstream effector molecules include p70 S6 ribosomalprotein kinase (S6K) and eukaryotic initiation factor binding inhibitoryprotein (4E-BP1). After the activation mTOR phosphorylates and activatesthe catalytic activity S6K1. mTOR also catalyzes phosphorylation of4E-BP1 and inactivates it, resulting in initiation of proteintranslation and cell cycle progression (Kozma et al, (2002) Bioessays24: 65). More importantly, mTOR exerts a negative feedback on activationof PI3K/AKT by suppressing expression and activation of IRS proteins.Inhibition of mTOR by rapamycin relieves the negative inhibition leadingto activation of PI3K AKT (Shi et al (2005) Mol Cancer Ther 2005; 4(10):1533).

PI3K/AKT/mTOR signaling pathway inhibition is considered a promisingcancer treatment (Engelman, J A, (2009) Nature Reviews: Cancer 9:551).mTOR antagonist rapamycin is the first signaling target in thePI3K/AKT/mTOR pathway for anti-cancer treatment (Courtney et al, (2010)J Clin Oncol 28: 1075-1083; Vivanco et al, (2002) Nat Rev Cancer2:489-50). Unfortunately, rapamycin lifts the negative feedbackinhibition of IRS proteins, leading to the activation of PI3K and AKT.Patients treated by rapamycin show increased AKT phosphorylation intumors, leading to failure of tumor treatment (Easton et al, (2006)Cancer Cell. 9 (3) :153-5) Therefore, there is a need to develop meansto effectively inhibit the PI3K/AKT/mTOR signaling pathway, inparticular to prevent the feedback activation of IRS proteins uponinhibition of mTOR.

SUMMARY OF THE INVENTION

The present invention relates to pharmaceutical agents that can inhibitPI3K/AKT and prevent mTOR antagonists (for example, rapamycin) inducedfeedback activation of PI3K and AKT, and such agents alone or incombination with mTOR antagonists will be used to treat PI3K/AKT/mTORrelated diseases. Accordingly, the present invention also relates to themethod and use of the pharmaceutical composition for the treatment andprevention of PI3K/AKT/mTOR-related diseases, in one aspect, the presentinvention includes introducing to patient an effective amount of PRCPantagonist, PREP antagonist, or an effective amount of PRCP and PREPdual antagonist for inhibition of PI3K/AKT/mTOR signaling pathway,thereby treating or preventing cancer and diseases caused by abnormalactivation of PI3K/AKT/mTOR signaling pathway. Antagonists include, butare not limited to the use of chemical inhibitors or inhibitorynucleotides. In another aspect, the invention also uses PRCPantagonists, PREP antagonists or dual antagonist PREP and PRCP toprevent feedback activation of PI3K/AKT by mTOR antagonists.

A. To use the effective dose by combining PRCP antagonists and mTORantagonists, or joint use of effective dose PREP antagonists and mTORantagonists, or a combination of the effective dose of PRCP PREP dualantagonists and mTOR antagonists to inhibit PI3K/AKT/mTOR signalingpathway, thereby treating or preventing abnormal activation of thePI3K/AKT/mTOR pathway related diseases. Antagonists include, but are notlimited to the use of chemical inhibitors or inhibitory nucleotides.Furthermore, the present invention also relates to the use of PRCPantagonists, PREP antagonists or dual antagonist PREP PRCP to destroyIRS proteins by using an effective amount of an antagonist for theaforementioned degradation of IRS protein, thereby treating orpreventing diseases related to IRS proteins, including the PI3K/AKT/mTORactivation related diseases, especially cancer. The aforementionedinvention, PRCP antagonist, PREP antagonists, or PREP and PRCPantagonists can be any dual antagonist. For example, they may beinhibitory nucleotides. Preferably, PRCP PREP antagonists and smallmolecule antagonists, and derivatives thereof; More preferably, thesmall-molecule compound is(tert-butyl(2s)-2-{[(2s)-2-formylpyrrolidin-1-yl]carbonyl}pyrrolidine-1-carboxylate)(Z-Pro-Prolinal or ZPP) and derivatives, and small molecule compounds[2-[8-(dimethylamino)octylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone(Y29794) and derivatives thereof. Further, preferably the small moleculeantagonists of mTOR are rapamycin and derivatives thereof. Wherein,preferably PI3K/AKT/mTOR abnormal activation of the signal pathwayassociated disease is cancer, neurodegenerative diseases, metabolicdiseases, hamartoma syndrome and hereditary myopathy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D. ZPP((tert-butyl(2s)-2-{[(2s)-2-formylpyrrolidin-1-yl]carbonyl}pyrrolidine-1-carboxylate))induces cytotoxicity in cancer cells analyzed by MTT assay and colonyformation assay (clonogenesis assay). FIG. 1A reports relative cellviability for cell lines Panc-1, AsPC-1, PK-9 and Capan-1 treated withZPP at various concentrations. FIG. 1B reports relative cell viabilityfor cell lines A549 and A1703 treated with various concentrations ofZPP. FIG. 1C reports relative cell viability for cell lines T47D andMCF7. FIG. 1D is a colony forming analysis for Panc-1 cell line treatedwith various concentrations of ZPP.

FIG. 2A-2C. Y29794([2-[8-(dimethylamino)octylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone)induces cytotoxicity in cancer cells analyzed by MTT assay. FIG. 2Areports relative cell viability for cell lines Panc-1 AsPC-1, and PK-9cells treated with various concentrations of Y29794. FIG. 2B reportsrelative cell viability for cell lines A549 and A1703 treated withvarious concentrations of Y29794. FIG. 2C reports relative cellviability for cell lines MDAMB231, T47D and MCF7 treated with variousconcentrations of Y29794.

FIG. 3. Gene silencing of PRCP or PREP individually by lentiviral shRNAreduces proliferation while gene silencing of both PRCP and PREP blockscell proliferation in Panc-1 pancreatic cancer cells. FIG. 3A reportsreduced proliferation of Panc-1 cells with knockdown of PRCP and/or PREPcompared with control cells. FIG. 3B reports reduction of PREP proteinin the cells with knockdown of PREP. FIG. 3C reports reduction of PREPprotein in the cells with knockdown of PRCP. KD1, KD2—knockdown 1 or 2of corresponding genes; DKD—double knockdown of PRCP and PREP.

FIG. 4. Gene silencing of PRCP or PREP individually by lentiviral shRNAreduces proliferation while gene silencing of both PRCP and PREP blockscell proliferation in PK-9 pancreatic cancer cells. FIG. 4A reportsreduced proliferation of PK-9 cells with knockdown of PRCP and/or PREPcompared with control cells. FIG. 4B reports reduction of PRCP and/orPREP mRNA in the cells with knockdown of PRCP and/or PREP, KD—knockdownof corresponding genes; DKD—double knockdown of PRCP and PREP.

FIG. 5. Gene silencing of PRCP or PREP individually by lentiviral shRNAreduces proliferation while gene silencing of both PRCP and PREP blockscell proliferation in Capan-1 pancreatic cancer cells. FIG. 5A reportsreduced proliferation of Capan-1 cells with knockdown of PRCP and/orPREP compared with control cells. FIG. 5B reports reduction of PRCPand/or PREP mRNA in the cells with knockdown of PRCP and/or PREP.KD—knockdown of corresponding genes; DKD—double knockdown of PRCP andPREP.

FIG. 6. Gene silencing of PRCP or PREP individually by lentiviral shRNAreduces proliferation while gene silencing of both PRCP and PREP blockscell proliferation in MCF7 breast cancer cells. FIG. 6A reports reducedproliferation of MCF7 cells with knockdown of PRCP and/or PREP. FIG. 6Breports reduction of PRCP protein in the cells with knockdown of PRCP.FIG. 6C reports reduction of PREP protein in the cells with knockdown ofPREP. FIG. 6D reports reduction of PRCP and PREP protein in the cellswith knockdown of PRCP and PREP. KD1, KD2—knockdown of correspondinggenes with two different pairs of shRNA (#1 or #2); DKD—double knockdownof PRCP and PREP.

FIG. 7 AKT phosphorylation is inhibited by gene silencing of PRCP and/orPREP in Panc-1, PK-9, Capan-1 and MCF7 cells by immunoblot analysis.FIG. 7A reports reduced AKT phosphorylation (S473) in Panc-1 cells withknockdown of PRCP and/or PREP. FIG. 7B reports reduced AKTphosphorylation (S473) in Capan-1 cells with knockdown of PRCP and/orPREP. FIG. 7B reports reduced AKT phosphorylation (S473) in Panc-1 cellswith knockdown of PRCP and/or PREP. FIG. 7D reports reduced AKTphosphorylation (S473) in MCF7 cells with knockdown of PRCP and PREP.KD1, KD2—knockdown of corresponding genes with shRNA #1; DKD—doubleknockdown of PRCP and PREP.

FIG. 8. AKT phosphorylation is inhibited by ZPP or Y29794 in Panc-1,PK-9, and MCF7 cells by immunoblot analysis. FIG. 8A reports reduced AKTphosphorylation (S473) in PK-9 cells treated with ZPP (400 μM) comparedwith cells treated with vehicle (DMSO). FIG. 8B reports reduced AKTphosphorylation (S473) in MCF7 cells treated with ZPP (400 μM) comparedwith cells treated with vehicle (DMSO). FIG. 8C reports reduced AKTphosphorylation (S473) in Panc-1 cells treated with ZPP (400 μM)compared with cells treated with vehicle (DMSO). FIG. 8D reports reducedAKT phosphorylation (S473) in Panc-1 cells treated with Y29794 (1 μM)compared with cells treated with vehicle (Ethanol). FIG. 8E reportsreduced AKT phosphorylation (S473) in MCF7 cells treated with Y29794 (1μM) compared with cells treated with vehicle (Ethanol). FIG. 8F reportsreduced AKT phosphorylation (S473) in PK-9 cells treated with Y29794 (1μM) compared with cells treated with vehicle (Ethanol).

FIG. 9. Immunoblot analysis of IRS-1 expression and AKT phosphorylationin Panc-1 cells: figure reports that expression of IRS-1 is reduced andphosphorylation of AKT is inhibited by gene silencing of PRCP and PREP.FIG. 9 also reports that increase in rapamycin-induced feedback in IRS-1expression and AKT phosphorylation is inhibited by gene silencing ofPRCP and PREP in Panc-1 cells. KD—knockdown of corresponding genes withshRNA #1; DKD—double knockdown of PRCP and PREP.

FIG. 10. ZPP reduces rapamycin-induced feedback increase in IRS-1 andAKT phosphorylation in Panc-1 and MCF cells by immunoblot analysis. FIG.10A reports that Panc-1 cells treated with rapamycin (10 nM) haveincreased IRS-1 protein, tyrosine phosphorylation of IRS-1, andphosphorylated AKT (S473), and that Panc-1 cells treated with ZPP (400μM) have reduced IRS-1 proteins and phosphorylated AKT (S473), while incells treated with ZPP and rapamycin, ZPP blocks rapamycin-inducedincrease in IRS-1 protein and phosphorylated AKT (S473). FIG. 10Breports that MCF7 cells treated with rapamycin (10 nM) have increasedIRS-1 protein and phosphorylated AKT (S473) and that MCF7 cells treatedwith ZPP (400 μM) have reduced IRS-1 proteins and phosphorylated AKT(S473), while in cells treated with ZPP and rapamycin, ZPP blocksrapamycin induced increase in IRS-1 protein and phosphorylated AKT(S473).

FIG. 11. Y29794 reduces rapamycin-induced feedback increase in IRS-1 andAKT phosphorylation in Panc-1 and MCF cells by immunoblot analysis. FIG.11A reports that Panc-1 cells treated with rapamycin (10 nM) haveincreased IRS-1 protein and phosphorylated AKT (S473) and that Panc-1cells treated with Y29794 (0.5 μM) have reduced phosphorylated AKT(S473), while the cells treated with Y29794 and rapamycin, Y29794 blocksrapamycin induced increase in IRS-1 protein and phosphorylated AKT(S473). FIG. 11B reports that MCF7 cells treated with rapamycin (10 nM)have increased IRS-1 protein and phosphorylated AKT (S473), while incells treated with Y29794 and rapamycin, Y29794 blocks rapamycin inducedincrease in IRS-1 protein and phosphorylated AKT (S473).

FIG. 12. FIG. 12 reports that Panc-1 cells treated with rapamycin haveincreased PI3K activity compared with vehicle-(DMSO)-treated cells byPI3K kinase assay in anti-p85 immunoprecipitates. FIG. 12 also reportsthat Panc-1 cells treated with ZPP or Y29794 have reduced PI3K activity,and that ZPP or Y29794 also blocks rapamycin-induced feedback increasein PI3K activity.

FIG. 13. FIG. 13 reports that Panc-1 cells treated with ZPP (400 μM) orY29794 (0.5 μM) have reduce PI3K activity compared with vehicle(DMSO)-treated cells by PI3K kinase assay of anti-IRS-1immunoprecipitates. FIG. 13 also reports that ZPP or Y29794 also reducestyrosine phosphorylation of IRS-1 and coimmunoprecipitation of p85 withIRS-1.

FIG. 14. Gene silencing of PRCP and PREP reduces PI3K activity andblocks rapamycin-induced feedback increase in PI3K activity. FIG. 14Areports that in Panc-1 cells rapamycin increases IRS-1 protein and IRS-1associated PI3K activity, and that in Panc-1 cells with double knockdownof PRCP and PREP, both IRS-1 protein and IRS-1 associated PI3K activityare reduced, and that this reduction is not reversed by rapamycin asshown by PI3K kinase assay in anti-IRS-1 immunoprecipitates (IP). FIG.14B reports that in Panc-1 cells rapamycin increases p85-associated PI3Kactivity, and that in Panc-1 cells with double knockdown of PRCP andPREP p85-associated PI3K activity is reduced, and that this reduction isnot reversed by rapamycin as shown by PI3K kinase assay in p85 IP.DKD—double knockdown of PRCP and PREP.

FIG. 15. Combination of ZPP and rapamycin induces synergisticcytotoxicity by MTT assay and colony formation assay in Panc-1 cells.FIG. 15A reports that Panc-1 cells treated with various concentration ofZPP in combination with 0.5 nM of rapamycin for three days havesynergistic reduction in cell viability. FIG. 15B reports that Panc-1cells treated with various concentration of rapamycin in combinationwith 25 μM of ZPP for three days have synergistic reduction in cellviability. FIG. 15C reports that Panc-1 cells treated with variousconcentration of rapamycin in combination with 50 μM of ZPP for 7 dayshave synergistic reduction in cell colonies formed at 21 days.

FIG. 16. Combination of Y29794 and rapamycin induces synergisticcytotoxicity by MTT assay and colony formation assay in Panc-1 cells.FIG. 16A reports that Panc-1 cells treated with various concentration ofY29794 in combination with 0.5 nM of rapamycin for three days havesynergistic reduction in cell viability. FIG. 16B reports that Panc-1cells treated with various concentration of rapamycin in combinationwith 25 nM of Y29794 for three days have synergistic reduction in cellviability. FIG. 16C reports that Panc-1 cells treated with variousconcentration of rapamycin in combination with 25 nM of Y29794 for 7days have synergistic reduction in cell colonies formed at 21 days.

FIG. 17. FIG. 17 reports that treatment of SCID mice withxenotransplanted Panc-1 tumor cells by Y29794 significantly reducesPanc-1 tumor growth; FIG. 17 also reports that treatment withcombination of rapamycin and Y29794 has synergistic effect in inhibitionof Panc-1 tumor growth.

FIG. 18. Gene silencing of PRCP and PREP induces serine phosphorylationand degradation of IRS-1 by immunoblot analysis. FIG. 18A reports thatin Panc-1, PK-9 and Capan-1 cells knockdown of PRCP and PREP reducesIRS-1 protein. FIG. 18B reports that in Panc-1 cells knockdown of PRCPand PREP induces serine phosphorylation (S307 and S636/639) of IRS-1while reduces tyrosine phosphorylation of IRS-1. FIG. 18C reports thatin Panc-1 cells knockdown of PRCP and PREP decreases IRS-1 half-lifewhen protein synthesis is blocked by cycloheximide (CHX, 20 μg/ml). FIG.18D reports that in control Panc-1 cells rapamycin inhibits serinephosphorylation (S307 and S636/639) and increases IRS-1 protein; whilein Panc-1 cells with knockdown of PRCP and PREP rapamycin does notinhibit S636/639 phosphorylation and does not increase IRS-1 protein.DKD—double knockdown of PREP and PREP.

FIG. 19. Y29794 induces serine phosphorylation and degradation of IRS-1and blocks rapamycin-induced feedback increase in IRS-1. FIG. 19Areports that in Panc-1. PK-9 and Capan-1 cells Y29794 (1 μM) reducesIRS-1 protein. FIG. 19B reports that in Panc-1 cells Y29794 (1 μM)induces serine phosphorylation (S307 and S636/639) of IRS-1 and reducesIRS-1 protein. FIG. 19C reports that in Panc-1 cells Y29794 (1 μM)decreases IRS-1 half-life in the presence of CHX. FIG. 19D reports thatin Panc-1 cells rapamycin induces increased IRS-1 protein level andinhibits mTOR phosphorylation, while Y29794 (1 μM) inhibitsrepamycin-induced increase in IRS-1 protein without affecting mTORphosphorylation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention, PRCP (gene library designation number (accessionnumber NP-005031, which isoforms (isoform) 1 is NP-005031.1; isoform 2is NP-955450.2) refers to a part of prolyl peptidase family and thefamily of carboxypeptidase serine peptidase. PRCP human amino acidsequence is shown in SEQ ID No. 1. PRCP cleaves the C-terminal prolinepeptide bond in the peptide. The present invention, PREP (gene libraryspecified number NP-002717) belongs to the prolyl peptidase family. PREPhuman amino acid sequence is shown in SEQ ID No. 2. Phylogeneticanalysis shows that PRCP and PREP contain highly similar amino acidsequences, and have a similar enzyme function. PRCP and PPEP can cleavesubstrates such as neuropeptide angiotensin II/III (Ang ll/III) andα-melanocyte stimulating hormone (α-MSH). PREP additionally cleavesvasopressin (neurotensin) and gastrin-releasing hormone(gastrin-releasing hormone) and other neuropeptides. These neuropeptidesactivate G protein-coupled receptor (GPCR) and regulate receptortyrosine kinase signaling pathway function through the G protein-coupledreceptors (2007) Neutopeptides 41: 1-24; Rosenblum J S et al, (2003)Curr Opin Chem Biol., 7:496-504; Skidgel et al, (1998) ImmunologicalReviews, 161: 129-41). Prolyl peptidase family includes acylaminoacylpeptidase (AAP), dipeptidyl-peptidases (DPP4, DPP7, DPP8, DPP9,fibroblast activation protein alpha (FAP)) (Rosenblum J S et al, (2003)Current Opinion in Chemical Biology, 7:496-504). PRCP is associated withobesity (Shariat-Madar B et al, (2010) Diabetes Metab Syndr Obes, 3:67-78). PRCP knockout mice was lower weight than the wild-type miceInhibition of PRCP enzyme function can also reduce mouse body weight.Our own research found that PRCP regulates cell proliferation, autophagyand resistance to tamoxifen in breast cancer cells (Duan L et al, (2011)JBC, 286:2864-2876). DPP4 is associated with obesity and diabetes. DPP4knockout mice fled with high-fat foods have lower body weight thanwild-type mice and are more sensitive to insulin (Richter B et al,(2008) Cochrane Database Syst Rev, 16; (2): CD006739). DPP4 inhibitorsare used to treat diabetes. DPP7 regulate the static lymphocytesurvival. FAP overexpression increased cell proliferation. PREP isassociated with amnesia, depression and Alzheimer's disease (Rosenblum JS et al, (2003) Current Opinion in Chemical Biology 7:496-504).

“Antagonist” used herein refers to the in vitro and in vivo agents thatcan reduce or prevent PRCP, PREP, and mTOR function. PRCP and PREPantagonists include but are not limited to, antagonists of otherproteins with similar functions within the carboxypeptidase family andthe prolyl peptidase family. mTOR antagonists include, but are notlimited to antagonists for other proteins involved in the activation ofthe mTOR signaling pathway. As used herein, the term refers to theinhibitory polynucleotide capable of inhibiting the expression of genes.Typical inhibitory polynucleotides include but not limited to antisenseoligonucleotides (Antisense oligonucleotides), triple helix DNA (triplehelix DNA), RNA aptamers (aptamers), ribozymes (ribozymes), shortinhibiting ribonucleotide (siRNA), short hairpin RNA (shRNA) andmicroRNA. For example, siRNA, microRNA, or antisense oligonucleotidesdesigned based on the known sequence of PRCP and PREP to inhibit theexpression of PRCP and PREP. Similarly, ribozymes can be synthesized torecognize specific nucleotide sequences of the gene and cut it. Theskilled in the art person is fully capable of using such prior artdesigns for gene inhibition without further development.

“Small molecule compounds” used herein refers to compounds with amolecular weight of less than 3 kilodaltons. A compound can be organicor a natural product.

“Effective dose” used herein refers to the dose which will affect thebiological function of the target molecule or signaling pathway, andthus can prevent or ameliorate clinical symptoms or condition. Theeffective dose is determined based on the intended goal. The effectivedose also refers to a dose that can reduce at least 10% of a targetmolecule or pathway activity or function in the host, preferably reduce30% or more preferably 50-90%.

“P13K/AKT/mTOR pathway-related diseases, P13K/AKT/mTOR signaling pathwayabnormalities caused by disease. PI3K/AKT/mTOR signal abnormalitiescaused by disease, PI3K/AKT/mTOR signal-related diseases and disorderscaused by signals of the same” used herein include, but are not limitedto, cancer, organ transplant-related disorders (for example, reduce therate of rejection, graft-versus-host disease, etc.), muscular sclerosis,arthritis, allergic encephalomyelitis, immunosuppression-relateddisorders, metabolic disorders (for example, obesity, diabetes, etc.),reducing intimal thickening after vascular injury, protein misfoldingdiseases (for example, Alzheimer's disease, Gaucher's disease,Parkinson's disease, Huntington's disease, cystic fibrosis, maculardegeneration, retinitis pigmentosa, diabetic retinopathy, prion disease,etc.). PI3K/AKT/mTOR signaling pathway-related diseases includehamartoma syndromes, such as tuberous sclerosis and multiple hamartomasyndrome. Hamartoma is a general term for benign tumor-like malformationcomposed of mature cells and tissue normally found in the affected areathat have grown in a disorganized manner.

PI3K/AKT/mTOR signaling pathway related disorders also includehereditary myopathy, myopathy such as myotubes. Myotubes myopathy ischaracterized by decrease in activity of muscle tubulin phosphatase(myotubularin). Myotubularin is a 3-phosphoinositide phosphatase.

“Cancer” is used herein to include mammalian solid tumors andhematologic malignancies, including but not limited to head and neckcancers lung cancer, pleural mesothelioma, esophageal cancer, gastriccancer, pancreatic cancer, hepatobiliary cancer, small bowel cancer,colon cancer, colorectal cancer, kidney cancer, urinary tract cancer,bladder cancer, prostate cancer, penile cancer, testicular cancer,gynecological cancer, ovarian cancer, breast cancer, endocrine systemcancer, skin cancer, CNS cancer, soft tissue sarcoma, osteosarcoma andmelanoma. Hematologic malignancies include but is not limited tolymphoma, multiple myeloma, Hodgkin's disease, leukemia, plasma celltumors and AIDS-related cancer. In addition, all of the other cancers,including primary cancer, metastatic cancer, in the context of recurrentcancer. Preferably the present invention for treating or preventingcancer and PI3K/AKT/mTOR abnormal activation of signaling pathwaysrelated to cancer diseases, neurodegenerative diseases, metabolicdiseases, hamartoma syndrome and hereditary myopathy. Preferably thepresent invention for treating or preventing cancer and abnormalactivation of PI3K/AKT/mTOR signaling pathway related to breast cancer,pancreatic cancer or lung cancer.

The present invention provides treating or preventing abnormalactivation of PI3K/AKT/mTOR signal pathway associated disorders usingpharmaceutical composition which comprises administering to a patient aneffective dose of ZPP and derivatives thereof; an effective amount ofY29794 and their derivatives; ZPP on combination with effective amountof rapamycin and its derivatives, and derivatives thereof; Y29794 incombination with effective amount of rapamycin and its derivativesthereof wherein the derivative is the compound of one or more chemicalreactions resulting in modification of the parent compounds withderivatives of the parent compounds having a similar structure, having asimilar effect on the function.

The invention also provides a method for treating or preventing abnormalactivation of PI3K/AKT/mTOR signal pathway associated diseases, whichcomprises administering to a patient an effective dose of an antagonistof PRCP, or an effective dose of PREP antagonist, and an effectiveamount of PRCP and PREP dual antagonists, and joint use of mTORantagonists with effective dose of PRCP antagonist, the joint use of aneffective dose PREP antagonists and mTOR antagonists, the joint use ofan effective dose of PRCP and PREP dual antagonists and an mTORantagonist. mTOR antagonists include antagonists of mTOR as well asantagonists of molecules directly upstream and downstream of mTOR signaltransduction.

PREP and PRCP chemical antagonists include(tert-butyl(2s)-2-{[(2s)-2-formylpyrrolidin-1-yl]carbonyl}pyrrolidine-1-carboxylate)(Z-Pro-Prolinal or ZPP), and derivatives thereof. In a particularembodiment, ZPP inhibits P13K kinase activity and phosphorylation ofAKT, ZPP also inhibits rapamycin-induced feedback activation of IRS-1,PI3K and AKT. ZPP is a prolinal compound. Currently ZPP and derivativesthereof as inhibitors and the production method is disclosed, forexample, reference to U.S. Pat. No. 5,411,976. ZPP derivatives include,but are not limited tobenzyl(2S)-2-[(2S)-2-formylpyrrolidine-1-carbonyl]pyrrolidine-1-carboxylate(Z-Pro-Pro-dimethyl acetal aldehyde); terephthalic acidbis(L-prolyl-pyrrolidine)amide;tert-butyl(2S)-2-(pyrrolidin-1-ylcarbonyl)pyrrolidine-1-carboxylate;UAMC -00021;4-phenyl-1-[(2S)-2-(pyrrolidine-1-carbonyl)pyrrolidin-1-yl]butan-1-one(SUAM 1221);((S)-2-[[(S)-2-(hydroxyacetyl)-1-pyrrolidinyl]carbonyl]-N-phenylmethyl)-1-pyrrolidinecarboxamide)(JTP-4819).

PREP chemical antagonists include[2-[8-(dimethylamino)octylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone(Y29794) and its derivatives. In one particular embodiment of thepresent invention, Y29794 induces IRS-1 serine phosphorylation anddegradation, inhibiting PI3K kinase activity and phosphorylation of AKT,Y29794 also inhibits rapamycin-induced feedback activation of IRS-1, PI3and AKT.

Y29794 is a pyridine compound. Currently Y29794 and derivatives thereofas inhibitors and the production method is disclosed, for example, inreference to U.S. Pat. No. 5,001,137. Y29794 derivatives include (butnot limited to)[2-[6-(dipropylamino)hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;[2-[6-(dimethylamino)hexylsulfanyl]-6-(2-methylpropyl)pyridin-3-yl]-thiophen-2-ylmethanone;[2-[6-(dimethylamino)hexylsulfanyl]-6-(3-methylbutyl)pyridin-3-yl]-thiophen-2-ylmethanone;[2-[6-(dimethylamino)hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-(5-methylthiophen-2-yl)methanone;[2-[6-(diethylamino)hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;[2-[6-(dimethylamino)hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;[2-[6-(dimethylamino)hexylsulfanyl]-6-propylpyridin-3-yl]-thiophen-2-ylmethanone;[2-[6-[2-(dimethylamino)ethyl-methylamino]hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;[2-[6-[benzyl(methyl)amino]hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;[6-tert-butyl-2-[6-(dimethylamino)hexylsulfanyl]pyridin-3-yl]-thiophen-2-ylmethanone;[2-[6-(dimethylamino)hexylsulfanyl]-6-methylpyridin-3-yl]-thiophen-2-ylmethanone;[2-[6-(dimethylamino)hexylsulfanyl]-4,6-dimethylpyridin-3-yl]-thiophen-2-ylmethanone;[2-[6-(butylamino)hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;[2-[6-(4-benzylpiperidin-1-yl)hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;[2-[8-(methylamino)octylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;[2-[6-(methylamino)hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;[2-[6-(tert-butylamino)hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;[6-propan-2-yl-2-[6-(propan-2-ylamino)hexylsulfanyl]pyridin-3-yl]-thiophen-2-ylmethanone;[2-[6-(ethylamino)hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;(E)-but-2-enedioic acid;[2-[6-(dimethylamino)hexylsulfanyl]-6-(3-methylbutyl)pyridin-3-yl]-thiophen-2-ylmethanone;[2-[6-(benzylamino)hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;[2-[6-(2-phenylethylamino)hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;[2-[8-(dimethylamino)octylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;oxalic acid;[2-[6-(dimethylamino)hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;oxalic acid;[2-[6-(diethylamino)hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;oxalic acid;[2-[6-(dimethylamino)hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-(5-methylthiophen-2-yl)methanone;oxalic acid;[2-[6-(dimethylamino)hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-phenylmethanone;[2-[6-(dimethylamino)hexylsulfanyl]-6-propylpyridin-3-yl]-thiophen-2-ylmethanone;oxalic acid; N,N-diethyl-2-methyl-6-thiophen-3-ylpyridine-3-carboxamide;N-(cyclopropylmethyl)-N,2-dimethyl-6-thiophen-3-ylpyridine-3-carboxamide;[6-propan-2-yl-2-[6-[4-[3-(trifluoromethyl)phenyl]piperazin-1-yl]hexylsulfanyl]pyridin-3-yl]-thiophen-2-ylmethanone;[2-[6-(dimethylamino)hexylsulfanyl]pyridin-3-yl]-(5-ethylthiophen-2-yl)methanone;[2-[6-[4-[bis(4-fluorophenyl)methyl]piperidin-1-yl]hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;[2-[6-[benzyl(methyl)amino]hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;oxalic acid;[6-tert-butyl-2-[6-(dimethylamino)hexylsulfanyl]pyridin-3-yl]-thiophen-2-ylmethanone;oxalic acid;[2-[6-(dimethylamino)hexylsulfanyl]-6-methylpyridin-3-yl]-phenylmethanone;[2-[8-(dimethylamino)octylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;4-methylbenzenesulfonic acid;[4-[(dimethylamino)methyl]piperidin-1-yl]-(2-methyl-6-thiophen-3-ylpyridin-3-yl)methanone;(2-methyl-6-thiophen-3-ylpyridin-3-yl)-piperidin-1-ylmethanone;N-(2-cyanopropyl)-N-ethyl-2-methyl-6-thiophen-3-ylpyridine-3-carboxamide;N,N,2-trimethyl-6-thiophen-3-ylpyridine-3-carboxamide;(2-methyl-6-thiophen-3-ylpyridin-3-yl)-thiomorpholin-4-ylmethanone;N,N,6-trimethyl-2-[1-(thiophen-3-ylmethyl)piperidin-4-yl]pyridine-3-carboxamide;[2-[1-[2-[bis(4-fluorophenyl)methyl]piperidin-1-yl]hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;[2-[5-(dimethylamino)pentylsulfanyl]pyridin-3-yl]-thiophen-2-ylmethanone;[2-[6-(butylamino)hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;oxalic acid;[2-[6-(ethylamino)hexylsulfanyl]-6-propan-2-ylpyridin-3-yl]-thiophen-2-ylmethanone;oxalic acid;[2-[6-(dipropylamino)hexylsulfanyl]pyridin-3-yl]-thiophen-2-ylmethanone;[2-[1-[6-(dimethylamino)hexylsulfanyl]-2-methylpropan-2-yl]sulfanylpyridin-3-yl]-thiophen-2-ylmethanone;[2-[8-(dimethylamino)octan-2-ylsulfanyl]pyridin-3-yl]-thiophen-2-ylmethanone;[2-[7-(dimethylamino)heptan-2-ylsulfanyl]pyridin-3-yl]-thiophen-2-ylmethanone;[2-[6-(dimethylamino)hexan-2-ylsulfanyl]pyridin-3-yl]-thiophen-2-ylmethanone;[2-[7-(dimethylamino)heptylsulfanyl]pyridin-3-yl]-thiophen-2-ylmethanone;[2-[2-[3-(dimethylamino)propylsulfanyl]ethylsulfanyl]pyridin-3-yl]-thiophen-2-ylmethanone;[2-[1-[3-(dimethylamino)propylsulfanyl]propan-2-ylsulfanyl]pyridin-3-yl]-thiophen-2-ylmethanone;[2-[2-[6-(dimethylamino)hexylsulfanyl]ethylsulfanyl]pyridin-3-yl]-thiophen-2-ylmethanone;[2-[1-[6-(dimethylamino)hexylsulfanyl]propan-2-ylsulfanyl]pyridin-3-yl]-thiophen-2-ylmethanone;[2-[6-(dimethylamino)hexylsulfanyl]pyridin-3-yl]-thiophen-2-ylmethanone.

mTOR chemical antagonists include rapamycin and rapamycin derivatives.Rapamycin (also sirolimus) is a known macrolide, its chemical name is(3S,6R,7E,9R,10R,12R,14S,15E,17E,19E,21S,23S,26R,27R,34aS)-9,10,12,13,14,21,22,23,24,25,26,27,32,33,34,34a-hexadecahydro-9,27-dihydroxy-3-[(1R)-2-[(1S,3R,4R)-4-hydroxy-3-methoxycyclohexyl]-1-methylethyl]-10,21-dimethoxy-6,8,12,14,20,26-hexamethyl-23,27-epoxy-3H-pyrido[2,1-c][1,4]oxaazacyclohentriacontine-1,5,11,28,29 (4H,6H,31H)-pentone.mTOR antagonists also include rapamycin derivatives. Rapamycin and itsderivatives as antagonists of mTOR chemistry are disclosed, for example,in reference to U.S. Pat. No. 3,993,749, U.S. Pat. No. 6,277,983, U.S.Pat. No. 7,384,953, Chinese Patent No. 200980154352.X. Many derivativesof rapamycin are known in the art. Rapamycin derivative examplesinclude, but are not limited to, everolimus, tacrolimus, CCI-779,ABT-578, AP-23675, AP-23573 AP-2384 7-Table-rapamycin Su 7-thiomethylrapamycin 7-Table-trimethoxyphenyl-rapamycin7-Table-thiomethyl-rapamycin 7-to methoxy-rapamycin ADM 32-tomethoxy-rapamycin 2-demethylation-rapamycin, before rapamycin(prerapamycin), temsirolimus matter (temsirolimus) and42-0-(2-hydroxy)ethyl-rapamycin. Other derivatives of rapamycin include:oximes of rapamycin (U.S. Pat. No. 5,446,048), the amino esters ofrapamycin (U.S. Pat. No. 5,130,307), two rapamycin aldehyde (U.S. Pat.No. 6,680,330), rapamycin 29-enolase (U.S. Pat. No. 6,677,357), 0-Dykeglycosylation (U.S. Pat. No. 6,440,990 rapamycin derivatives),water-soluble esters of rapamycin (U.S. Pat. No. 5,955,457), alkyl withrapamycin derivatives (U.S. Pat. No. 5,922,730), amidino carbamates ofrapamycin (U.S. Pat. No. 5,637,590), rapamycin biotin esters (U.S. Pat.No. 5,504,091), carbamates of rapamycin (U.S. Pat. No. 5,567,709), thehydroxy ester of rapamycin (U.S. Pat. No. 5,362,718), rapamycin42-sulfonate and 42-(N-oxy-chi Dyke) amino acid esters (U.S. Pat. No.5,346,893), rapamycin epoxycyclohexane alkyl isomers (U.S. Pat. No.5,344,833), imidazolidine derivatives of rapamycin (U.S. Pat. No.5,310,903), alkoxyalkyl esters of rapamycin (U.S. Pat. No. 5,233,036),pyrazole rapamycin (U.S. Pat. No. 5,164,399), acyl derivatives ofrapamycin (U.S. Pat. No. 4,316,885), the reduction product of rapamycin(U.S. Pat. No. 5,102,876 and U.S. Pat. No. 5,138,051), amide esters ofrapamycin (U.S. Pat. No. 5,118,667), fluorinated esters of rapamycinU.S. Pat. No. 5,100,883), acetal rapamycin (U.S. Pat. No. 5,151,413),oxa-rapamycin (U.S. Pat. No. 6,399,625), and silyl ethers of rapamycin(U.S. Pat. No. 5,120,842).

The present invention is a pharmaceutical composition comprisingrapamycin and its derivatives, and ZPP and derivatives thereof. Anotherpresent invention is a pharmaceutical composition comprising rapamycinand derivatives thereof, and Y29794 and derivatives thereof.

Below with reference to specific embodiments, the present invention willbe further elaborated. The following examples illustrate preferredembodiments of the present invention. The skilled person will appreciatethat, in the embodiment of the present invention showed good techniquedisclosed embodiment represent techniques discovered by the inventors inthe following examples, and therefore it can be considered to constitutepreferred modes. However, it should be understood that these examplesare intended to illustrate the invention and not to limit the scope ofthe invention. According to the present specification, the skilledperson will understand that many modifications and changes may be madeto the present invention without departing from the spirit of the scopeof the disclosed embodiment, and still obtain a like or similar result.A person skilled in the art understands the conventional methoddescribed in the experimental method described below, if no specialinstructions are presented or materials used; if no special instructionsare presented, routine biochemical reagents were purchased. In aparticular embodiment, PRCP and PREP plays a necessary role inPI3K/AKT/mTOR signaling activation and proliferation and survival ofcancer cells.

By including breast cancer cell lines MCF7 and TD47, pancreatic cancercell line Panc-1, PK-9, Capan-1 and AsPC-1, and lung cancer cell linesA549 and H1703 presented results indicate that gene silencing of PREP orPRCP decreases proliferation of cancer cells, and dual silencing of geneexpression PREP and PRCP causes proliferation arrest of cancer cells.The present invention indicates that specifically inhibiting PRCP orPREP gene expression, dual inhibition of gene expression PREP and PRCPreduce IRS-1, PI3K and AKT activity, and prevents rapamycin-inducedfeedback activation of IRS-1, PI3K and AKT. In another particularembodiment, the inhibitory effect of ZPP and Y29794 on PI3K/AKT/mTORsignaling pathway and cancer cell proliferation and survival show thatZPP or Y29794 stop the proliferation of cancer cells. ZPP or Y29794reduce insulin receptor substrate (IRS-1) protein, thereby inhibitingPI3K and AKT activity. ZPP or Y29794 prevents rapamycin-induced feedbackincrease in insulin receptor substrate (IRS-1), thereby inhibiting theactivity of PI3K and AKT. And, ZPP or Y29794 in combination withrapamycin together have a synergistic effect on inhibition of cancercell proliferation and survival. Y29794 inhibits pancreatic tumor growthin tumor xenograft experiments in immunodeficient mice. Y29794 incombination with rapamycin has synergistic effect in inhibition of tumorgrowth.

EXAMPLE 1 ZPP and Y29794 Induces Cytotoxicity in Cancer Cells

-   -   1.1. ZPP and Y29794 induces cytotoxicity to cancer cells by MTT        analysis of cell viability. Cells were placed in 96-well plates        (3×10 cells/well) in octuplicate. The cells were then treated        with vehicle or different doses of ZPP for 4 days. Cells were        then loaded with 1.2 mM MTT        (4,5-Dimethylthiazol-2-vn-2,5-diphenyltetrazolium bromide, a        yellow tetrazole) in phenol red-free medium for 4 hours. The        cells were then lysed in 10% SDS/0.01M HCL. MTT absorbance (570        nm spectrum) was measured by a microplate reader. The relative        absorbance is normalized to the control (vehicle-treated) to        indicate relative cell viability. ZPP was purchased from Biomol        (Plymouth Meeting, Pa., USA). MTT was purchased from        Sigma-Aldrich. Panc-1 and Capan-1 pancreatic cancer cell line,        MCF7 and T47D breast cancer cell lines, A549 and HI 703 lung        cancer Cell lines were purchased from American Type Culture        Collection (ATCC, USA) preparation of pancreatic cancer cell        line PK-9 have been disclosed, e.g., Kobari M et al, (1986)        Tohoku J Exp Med. 150: 231-248; Etoh T et al, (2003) Clin Cancer        Res 9; 1218; Arafat H. et al, (2011) Surgery 150 (2): 306-315.        Cells were grown in Dulbecco's Modified. Eagle's (DMEM) medium        (Invitrogen, Carlsbad, Calif.) containing 10% fetal bovine serum        (FBS) (Hyclone, purchased from Thermo Fisher Scientific, Inc).        Results indicate that ZPP decreases MTT absorbance in a dose        dependent manner in pancreatic cancer cell lines Panc-1, PK-9,        Capan-1 and AsPC-1 (FIG. 1A), lung cancer cell lines H1703 and        A549 (FIG. 1B), and breast cancer cell lines MCF7 and TD47 (FIG.        1C), indicating ZPP is cytotoxic to cancer cells. In similar        experiments, the cells were treated with different doses of        Y29794 for 4 days and analyzed for MTT absorbance. Y29794        oxalate was purchased from Tocris Biosciences (Bristol, UK). The        results show that in pancreatic cancer cell lines Panc-1, PK-9,        Capan-1 and AsPC-1 (FIG. 2A), lung cancer cell line H1703 and        A549 (FIG. 2B), and breast cancer cell lines MCF7, TD47 and        MDAMB231 (FIG. 2C), Y29794 shows a dose-dependent cytotoxicity        to cancer cells.    -   1.2. Colony formation assay (clonogenic assay) was used to        analyze the effect of ZPP or Y29794 on cancer cell survival. 10³        Panc-1 cells were plated in 100 mm petri dish in triplicate fed        with 10 ml of DMEM containing 10% FBS. The cells were then        treated by ZPP for 7 days and cultured for additional three        weeks. Cells were then rinsed three times with PBS, fixed with        100% ethanol for 15min, and stained with 2% crystal violet        ethanol solution. The stained colonies were counted. All the        results are statistically analyzed by one way ANOVA and two        tailed t-test. The results showed that ZPP significantly        (P<0.01) decreased Panc-1 pancreatic cancer cell survival (FIG.        1D).

EXAMPLE 2 PRCP and/or PREP Gene Silencing Inhibits Cancer CellProliferation

2.1. Lentiviral shRNA Silencing of PREP and/or PRCP Genes in CancerCells

Lentiviral vector (pLKO. 1) with PREP shRNA (PREP shRNA #1 cloneidentification number TRCN0000050198; PREP shRNA #2 clone identificationnumber TRC0000050199) and PRCP shRNA (PRCP shRNA #1 clone identificationnumber TRCN0000050808; PRCP shRNA #2 clone identification numberTRC0000050809) were purchased from OpenBiosystems (USA). pLKO. 1 withshRNA plasmids and viral packaging vectors psPAX2 and pMD2G (purchasedfrom Addgene (Cambridge, Mass., USA) plasmids were used for PRCP andPREP gene silencing. Viral packaging cells 293FT were purchased fromInvitrogen Corporation (Carlsbad, Calif., USA). Specific methods: (1)Generating viral supernatant of PREP shRNA or PRCP shRNA: 5×10⁵ 293FTviral packaging cells were placed in 100 mm petri dish in 10 ml of DMEMcontaining 10% FBS. The next day, 6 micrograms of plasmid of PRCP shRNA(or PREP shRNA), 3 micrograms of the psPAX2 plasmid, and 6 micrograms ofpMD2.G plasmid DNA were mixed in 500 microliters of culture medium with45 microliters of Fugene transfection reagent (Promega Corp, Madison,Wis., USA) for 15 minutes. This mixture was then added to the 293FTpackaging cells. On the third day, the supernatant containing viruseswas collected and filtered through a 0.45 micron syringe filter; (2)viral infection of the experimental cells: 5×10⁵ experimental cells(e.g., pancreatic carcinoma cell line Panc-1, PK-9, Capan-1, and MCF7breast cancer cell lines, etc.) were placed in 100 mm petri dishes. 5 mlviral supernatant was mixed with 5 ml culture medium and added to thecells. After 24 hours, puromycin (1 μg/ml) was added to the medium forselection of the infected cells. One week after selection the cells wereused for further experiments.

2.2 Immunoblot analysis of gene silencing of PRCP or PREP in cancercells. Mouse anti-PRCP antibody was purchased from Abeam (Cambridge,Mass., USA). Goat anti-PREP antibody was purchased from R&D Systems(Minneapolis, Minn., USA). Mouse anti-β-actin antibody antibodies wasfrom Santa Cruz Biotechnology (Santa Cruz, Calif., USA). Panc-1pancreatic cancer cells and MCF7 breast cancer cells were cultured to80% confluence, cells were rinsed three times with pre-cooled PBS, 500μl of lysis buffer (150 mmol/L Nacl, 1% Triton-x-100, 50 mmol/l Tris pH8.0, 1 mmol/L PMSF, 1 ug/L aprotinin. 1 ug/L leupeptin, 1 ug/L peptain)was added to the cells. Cells were scraped and transferred to acentrifuge tube with a pipette. The lysates were spun in amicrocentrifuge at maximal speed for 10 minutes at 4° C. to get rid ofthe insoluble fraction. Protein concentration was determined by Bradfordassay. For immunoprecipitation: 2-5 ug protein-specific antibodies wereadded to 250-500 ug of lysates in a tube and incubated on an orditalshaker 4° C. for 2-4 hours with moderate shaking, then 20 ul Protein Gbeads (Santa Cruz Biotechnology (Santa Cruz, Calif., USA.) were addedand the mixture was incubated with moderate shaking for another hour.After washing the beads in lysis buffer five times, theimmunoprecipitates were boiled in 2× Laemmli sample buffer (50 mmoL/LTris-HCL (pH 8.0), 100 mmoL/L DTT, 2% SDS, 0.1% bromophenol blue, 10%glycerol) for five minutes. The proteins were separated by conventionalSDS-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis andtransferred to Amersham Hybond-P PVDF membrane (GE Company, Pittsburgh,Pa., USA). For immunoblot, the membrane was blocked in 2% BSA in TBSTbuffer (Tris 1.21 g NaC15.84 g+800 ml H₂O adjusted to pH 8.0 with HC1)for one hour and then incubated with primary antibodies in TBST at roomtemperature (22-25° C.) for 2 hours. After washing three times withTBST, the membrane was incubated with HRP-conjugated secondaryantibodies for 0.5 hours at room temperature. After washing the membranefive times with TBST minutes/each), the membrane was incubated with ECLchromogenic reagent (GE Company, Pittsburgh, Pa., USA) for 1-5 minutes.The blots were exposed to films in darkroom for 1-5 minute. The filmswere developed in an automatic X-OMAT Developer (KODAK, Rochester, N.Y.,USA). The results show that in the cells with stable silencing of PREP(PREP KD) or PRCP (PRCP KD) or both PREP and PRCP (DKD), expression ofPREP or PRCP proteins were reduced (FIGS. 3B and C). PREP gene silencing(PREP KD) or PRCP gene silencing (PRCP KD) or gene silencing both PRCPand PREP (DKD) in MCF7 cells reduces expression of PREP and/or PRCPproteins compared with control cells (control) (FIGS. 6B & C). Thus, inPanc-1 and MCF7 cells, PRCP gene silencing, PREP gene silencing or PRCPand PREP gene silencing successfully reduced expression of PRCP and/orPREP proteins in the cells.

2.3 Real-Time Quantitative PCR Analysis of PRCP or PREP Gene Expressionin Cancer Cells with PRCP and/or PREP Gene Silencing.

Trizol, superscript III first-strand synthesis supermix, SybrGreen qPCRsupermix was purchased from Invitrogen (Carlsbad, Calif., USA). PRCPprimers (Forward: TCTACACTGGTAATGAAGGGGAC (shown as SEQ ID No.3),reverse: TCCTTGAATGAGTTGTCACCAAA (shown as SEQ ID No.4)). PREP primers(forward: GAGACCGCCGTACAGGATTAT (shown as SEQ ID No.5), reverse:TGAAGTGGCAACTATACTTGGGA (shown as SEQ ID No.6) were synthesized byIntegrated DNA Technologies Company (Coralville, Iowa, USA). Specificmethods: (I) fotal RNA extraction: pancreatic cancer cells PK-9 andCapan-1 at 80% confluence were rinsed with ice-cold PBS three times,lysed in 1 ml Trizol at RT for 5 min and then mixed with 0.2 ml ofchloroform for 2 to 3 min. The mixture was centrifuged at 12000 g at 4°C. for 15 min. The supernatant (approximately 0.6 ml) was transferred toa new tube with addition of 0.6 ml chloroform and incubate at RT for 2min and then centrifuged for another 15 min (secondary chloroformextraction). Equal volume of isopropanol was added to the supernatant toprecipitate RNA by centrifugation at 12000 g for 10 min. Theprecipitates were washed with 1 ml 75% of DEPC-ETOH and centrifuged for5 min. RNA was dried and dissolved in water. RNA concentration andpurify was determined by measuring A₂₆₀/A₂₈₀ and A_(26o)/A₂₃₀ values,(2) First strand cDNA synthesis (superscript III supermix, ThermoFisher, Grand Island, N.Y., USA): 2XRT reaction mix 10 μl, RT enzyme mix2 RNA (1 μg), nuclease-free water, total volume 20 μl. Gently mix, 25°C. incubation 10 min, 37° C. incubation 120 min, 85° C. 5 min, and thenplaced on ice, (4) qPCR reaction (SybrGreen qPCR Master Mix, AppliedBiosystems, Warrington, UK)): Mixed SybrGreen supermix universal 2×10μl, forward primer (4 μM) in a PCR tube 1 μl, reverse primer (4 μM) 1μl, cDNA. (1 μg from total RNA) 1 μl, nuclease-free water to 20 μlreacted on real-time PCR instrument (Thermo Fisher, Grand Island, N.Y.,USA) with standard procedure: 50° C. 2 min, 95° C. 10 min, 40 cycle: 95°C. 15 s, 60° C. 60 s. Relative abundance of cDNA was calculated bystandard curve method. The PCR results showed that PRCP and/or PREP mRNAwas reduced in PK-9 cells (FIG. 4B) and Capan-1 cells (FIG. 5B) withgene silencing of PRCP (PRCP KD), PREP (PREP KD), or both PRCP and PREP(DKD) compared to control cells (control).

2.4 Cell proliferation assay by staining cellular DNA in cells with genesilencing of PRCP, PREP, and both of PREP and PRCP. Cells were placed in96-well plates (3×10³ cells/well) in octuplicate. All cells were grownin DMEM containing 10% FBS. Medium was refreshed every two days. Cellswere harvested at day one, day four and day seven by freezing andthawing in 100 μl of TE (pH 8) buffer after removing medium and rinsingwith PBS for three times, Cellular DNA was stained with Picogreen(Invitrogen, Grand Island, N.Y., USA) in 1:200 dilution in TE buffer for30 min, the fluorescence was measured by a microplate reader withexcitation/emission at 480 nm/520 nm). Relative fluorescence intensityof Picogreen (average from octuplicate) was normalized to cellsharvested at day 1 to indicate cell proliferation. Compared to controlcells (control), pancreatic cancer cell lines Panc-1 (FIG. 3A), PK-9(FIG. 4A), Capan-1 (FIG. 5A) and breast cancer cell line MCF7 (FIG. 6A)with gene silencing of PRCP (PRCP KD1 and PRCP KD2) and/or PREP (PREPKD1 and PREP KD2) show reduced proliferation.

EXAMPLE 3 Gene Silencing of PREP and PRCP Genes, ZPP or Y29794 TreatmentDecreases IRS-1, PI3K and AKT Activity and Blocks Rapamycin-InducedFeedback Activation of IRS-1 and AKT

3.1 Reduction in AKT phosphorylation is examined by immunoblot usingrabbit anti-phospho-AKT (S437) and rabbit anti-pan AKT antibodiespurchased from Cell Signaling Technology (Danvers, Mass., USA) in thecells with gene silencing of PRCP and/or PREP or treated with ZPP orY29794. Reduction in PRCP and/or PREP is shown by Western blot andquantitative PCR described in 2.2.

Immunoblot analysis of cell lysates of PK-9 (FIG. 7A), Capan-1 (FIG. 7B)and Panc-1 (FIG. 7C) by comparing control cells (control) to the cellswith PRCP gene silencing (PRCP-KD1) or PREP gene silencing (PREP-KD1) ordouble PRCP and PREP gene silencing (DKD) show that phosphorylated AKT(p-AKT) was significantly reduced in the cells with gene silencing ofPRCP and PREP. MCF7 cell lysates by immunoblot analysis (FIG. 7D) showthat phosphorylation of AKT is also significantly reduced by genesilencing of both PRCP and PREP (DKD) compared to control cells(control).

PK-9 (FIG. 8A), Capan-1 (FIG. 8B) and Panc-1 (FIG. 8C) were treated withZPP (200 μM) for two days. Immunoblot analysis of cell lysates showsthat AKT phosphorylation in ZPP treated cells is significantly decreasedcompared to vehicle (DMSO) treated cells. Panc-1 (FIG. 8D), MCF7 (FIG.8E) and PK-9 (FIG. 8F) were treated with Y29794 (0.5 μM) for two days.Immunoblot analysis of cell lysates shows that AKT phosphorylation inY29794 treated cells is significantly decreased compared to vehicle(ethanol) treated cells.

3.2 AKT phosphorylation was examined by immunoblot as described in 3.1.IRS-1 expression was analyzed by immunoblot using anti-IRS-1 antibodiespurchased from Santa Cruz Biotechnology (Santa Cruz, Calif., USA).Reduction in PRCP and/or PREP by gene silencing is shown by Western blotand quantitative PCR described in 2.2.

Panc-1 cells were treated with rapamycin (10 nM) for 24 hours and celllysates were immunoblotted for IRS-1 and phospho-AKT and AKT. In controlcells, rapamycin treatment significantly increased IRS-1. protein andAKT phosphorylation. In cells with silenced PRCP gene (PRCP KD (FIG.9)), or cells with silenced PREP gene (PREP KD (FIG. 9)), the increasein IRS-1 and phospho-AKT induced by rapamycin was slightly lower thanthe control cells. In cells with both PRCP and PREP genes silenced (DKD(FIG. 9)), rapamycin-induced increase in IRS-1 and phospho-AKT wassignificantly lower than that in control cells.

Panc-1 cells (FIG. 10A) and MCF7 (FIG. 10B) cells were treated withvehicle, rapamycin (10 nM), ZPP (200 μM) or rapamycin plus ZPP for 24hours. Cell lysates were immunoblotted for phospho-AKT, AKT, IRS-1, andβ-actin. Rapamycin induced an increase in IRS-1 and phosphorylation ofAKT, which was blocked by ZPP treatment. In another experiment, Panc-1cells (FIG. 11A) and MCF7 (FIG. 11B) cells were treated with vehicle,rapamycin (10 nM), Y29794 (0.5 μM) or rapamycin plus Y29794 for 24hours. Cell lysates were immunoblotted for phospho-AKT, AKT, IRS-1, andβ-actin. Rapamycin induced an increase in IRS-1 and phosphorylation ofAKT, which was blocked by Y29794 treatment.

3.3 PI3K activity as well as rapamycin-induced feedback increase in PI3Kactivity is inhibited by gene silencing of PRCP and PREP or treatmentwith ZPP or Y29794 by PI3K kinase assay

PI3K kinase assay kit (Calbiochem, Bilerica, Mass., USA) was used tomeasure the IRS-1-associated or p85-associated PI3K kinase activity incells. The indicated cells were treated with different conditions for 24hours. The cells were rinsed three times with pre-cooled PBS, lysed in500 μl of lysis buffer (150 mmol/L Nacl, % Triton-x-100, 50 mmol/1 Tris(pH8.0, 1 mmol/L PMSF, 1 μg/L aprotinin, 1 μg/L leupeptin, 1 μg/Lpeptain). Cell lysates (500 μg, in triplicate) of were used foranti-IRS-1 or anti-p85 immunoprecipitation. The immunoprecipitates wereresuspended in 200 μl assay buffer and mixed with thefluorescence-labeled PI3K substrate BODIPY-TMR-phosphatidylinositol (100μM) and ATP (37° C.) for 1 hour. The fluorescence intensity was measuredwith a fluorometer (excitation wavelength of 540 nM, emission wavelength570 nM). The difference between fluorescence intensity between controlimmunoprecipitates (no lysates) and lysate immunoprecipitates was usedto indicate PI3K kinase activity. IR-1 anti-rabbit antibody and rabbitanti-PI3K p85 antibody were purchased from Millipore Corporation(Billerica, Mass., USA). Panc-1 anti-p85 immunoprecipitates were usedfor PI3K kinase assay (FIG. 12) and immunoblot analysis (FIG. 12).Rapamycin treatment in control cells increased PI3K kinase activity(P<0.01). In cells treated with ZPP (200 μM) PI3K kinase activity wassignificantly reduced (P<0.01), and rapamycin-induced increased in PI3Kactivity was inhibited by ZPP (P<0.01); in cells treated with Y29794(0.5 μM) PI3K activity was significantly lower (P<0.01), Y29794 alsoinhibited rapamycin-.induced increase in PI3K kinase activity (P<0.01),anti-IRS-1 antibody (FIG. 14A) and anti-p85 PI3K antibody (FIG. 14B) wasused to analyze PI3K kinase activity in Panc-1 cell lysates (FIG. 14) ofin control cells and the cells with gene silencing of PRCP and PREP(DKD). In rapamycin treated control cells (control) the IRS-1 (FIG. 14A)and p85-associated PI3K activity was significantly elevated (FIG. 14B)(P<0.01). In the DKD cells IRS-1 (FIG. 14A) and p85 (FIG. 14B)associated PI3K activity were significantly reduced (P<0.01) compared tocontrol cells. Rapamycin failed to induce significant increase in PI3Kactivity in DKD cells (FIG. 14B) (P>0.5).

3.4 ZPP or Y29794 inhibits IRS-1 tyrosine phosphorylation and p85 PI3Ksubunit interaction with IRS-1 and PI3K kinase activity. Panc-1 cellswere treated with ZPP (200 μM) or Y29794 (0.5 μM) for 24 hours. Lysateswere immunoprecipitated with anti-IRS-1 antibodies and the anti-IRS-1immunoprecipitates were used for PI3K kinase assay and anti-IRS-1phospho-tyrosine and anti-p85 immunoblot analysis (FIG. 13). ZPP orY29794 decreased IRS-1-associated PI3K activity (FIG. 13, upper) andtyrosine phosphorylation of IRS-1 and p85 (FIG. 13, lower).

EXAMPLE 4 ZPP, Y29794, or its Combination with Rapamycin InducesSynergistic Cytotoxicity to Cancer Cells In Vitro

4.1 MTT Cell Viability Assay and Colony Formation Assay for Evaluationof Cytotoxic Effect of ZPP and its Combination with Rapamycin.

Examples 1.1 MTT cell viability assay experiments and examples 1.2colony-forming experiments to detect the combination of ZPP andrapamycin synergistically increased drug cytotoxicity. Panc-1 cells weretreated with different doses of ZPP alone or in combination withrapamycin (0.5 nM) (FIG. 15A), or with different doses of rapamycinalone or in combination with ZPP (25 μM) (FIG. 15B) for four days. MTTassay showed that rapamycin significantly increased (P<0.01) ZPP-inducedcytotoxicity (FIG. 15A), and vice versa, ZPP significantly (P<0.01)increased rapamycin-induced cytotoxicity (FIG. 15B). Colony formationassay (FIG. 15C) showed that rapamycin (1 nM and 10 nM) alone did notaffect cell survival. ZPP (25 μM) in combination with rapamycinsignificantly (P<0.01) decreased cell survival.

4.2 MTT Cell Viability Assay and Colony Formation Assay for Evaluationof Cytotoxic Effect of Y29794 and its Combination with Rapamycin.

Examples 1.1 MTT cell viability assay experiments and examples 1.2colony-forming experiments to detect the combination of Y29794 andrapamycin synergistically increased drug cytotoxicity. Panc-1 cells weretreated with different doses of Y29794 alone or in combination withrapamycin (0.5 nM) (FIG. 16A), or with different closes of rapamycinalone or in combination with Y29794 (25 nM) (FIG. 16B) for four days.MTT assay showed that rapamycin significantly increased (P<0.01)Y29794-induced cytotoxicity (FIG. 16A), and vice versa, Y29794significantly (P<0.01) increased rapamycin-induced cytotoxicity (FIG.16B). Colony formation assay (FIG. 16C) showed that rapamycin (1 nM and10 nM) alone did not affect cell survival. Y29794 (25 nM) combinationwith rapamycin significantly (P<0.01) decreased cell survival.

EXAMPLE 5 Y29794 Inhibits Pancreatic Tumor Growth and Combination ofY29794 with Rapamycin Synergistically Inhibits Tumor Growth

5:1 Analysis of Therapeutic Effect of Y29794 and its Combination withRapamycin in Xenotransplanted Pancreatic Tumor Growth.

4-6 week-old male SCID mice (BALB/C) were purchased from Charles Riverlaboratories (Wilmington, Mass., USA). Pane-1 cells at about 80%confluence were trypsinized with 0.25% trypsin and 0.02% EDTA. The cellsuspension was centrifuged after addition of 10% FBS. 10⁶ cells wereresuspended in 100 μl PBS buffer containing 10% Matrigel, and injectedwith a 1-cc syringe with a 27-30 sized needle into the right flank ofnude mice under anesthesia. After tumor formation (mean volume 50-60mm³) the mice were randomly divided into four groups (6 mice per group):control group, rapamycin treatment group, Y29794 treatment group, andY29794 plus rapamycin treatment group. Rapamycin and Y29794 weredissolved in Cremophor formulation (20% Cremophor EL, 30% propyleneglycol, 50% ethanol). Rapamycin (2 mg/kg body weight) was given byintraperitoneal injection one dose per week. Y29794 (20 mg/kg bodyweight) was administered by gavage five doses a week. Weeklymeasurements of tumor size (length, width and depth) and calculation ofthe tumor volume (1/2 larger diameter×(smaller diameter)) were carrieduntil the control tumors reached 1000 mm³. The results (FIG. 17) showedthat Y29794 alone significantly inhibited tumor growth (P<0.01).Combination of rapamycin with Y29794 inhibited tumor growth (P<0.01)more than Y29794 alone.

EXAMPLE 6 PRCP and PREP Gene Silencing or Y29794 Induced IRS-1 SerinePhosphorylation and Degradation by Immunoblot Analysis

As described in Example 2.2 and above, the cells with PRCP and PREP genesilencing were used for analysis of PRCP and PREP gene silencing andinhibition of serine phosphorylation and degradation of IRS-1 and itseffects on response to rapamycin. The antibodies anti-IRS-1, anti-p-IRS(S307), anti p-IRS-1 (S636/639), anti p-S6K (T389), Anti-S6K,anti-insulin receptor (IR), insulin resistance like growth factor-1receptor, anti-p-mTOR (S2448), anti-mTOR antibody was purchased fromCell Signaling (Denver, Mass., USA). The protein synthesis inhibitorcycloheximide (CHX) was purchased from Sigma Aldrich (TOWN, STATE, USA).Immunoblot analysis of cell lysates showed FIG. 18 that in cells withsilencing of both PRCP and PREP genes (DKD in PK-9, Capan-1 and Panc-1cells), insulin receptor substrate (IRS-1) protein was significantlyreduced compared to control cells (control), while insulin-like growthfactor receptor (IGF-1R) protein did not change (see FIG. 18A),indicating PRCP and PREP exclusively regulate IRS-1 but not IGF1Rprotein level. Compared to control cells, DKD cells show increasedserine phosphorylation (S307 and S636/639) of IRS-1 alongside withdecreased tyrosine phosphorylation of IRS-1 (FIG. 18B). To determinedegradation of IRS-1, the cells were treated with CHX (20 μg/ml) toblock protein synthesis for a time course. Immunoblot analysis oflysates showed that gene silencing of both PRCP and PREP genes shortenedthe half-life of IRS-1 compared to control cells, indicating that PRCPand PREP regulate the stability of IRS proteins (FIG. 18C). Whilerapamycin treatment stabilized IRS-1 in control cells by inhibitingserine 307 and serine 636/639 phosphorylation (FIG. 18C) silencing ofboth PRCP and PREP genes maintained phosphorylation of serine 636/639and decreased IRS-1 stability, while rapamycin completely inhibitedphosphorylation of S6K (T389) (FIG. 18D). The results indicate that PRCPand PREP inhibit serine phosphorylation and degradation of IRS-1.Similarly, inhibition of PREP/PRCP by Y29794 also induced serinephosphorylation and protein degradation of IRS-1 (see FIG. 19).Immunoblot analysis of Panc-1 cell lysates showed that, compared tocontrol cells, Y29794-treated cells had significantly reduced IRS-1protein (FIG. 19A) and increased serine phosphorylation (S307 andS636/639) of IRS-1 (FIG. 19B). Blocking protein synthesis by CHX incells treated with Y29794 showed that Y29794 shortened half-life ofIRS-1 (FIG. 19C) compared to vehicle treated cells (control). Moreover,while in control cells rapamycin induced increase in IRS-1 protein thatwas stable, in the cells treated with Y29794 IRS-1 protein was notincreased nor stabilized by rapamycin, although rapamycin completelyinhibited mTOR (S2448) phosphorylation.

Referred to herein and in any publication, references, patents andpatent applications shall be deemed incorporated by reference in theirentirety in this application, and should be regarded as based on a clearand independent way of reference in each individual publication,references, patents or patent applications. Any and all examples, orexemplary language provided herein (e.g., such as etc.) is intendedmerely to better illuminate the invention and does not form arestriction on the scope of the invention unless otherwise required. Thepresent invention describes preferred embodiments, including the bestmode known to the inventors for carrying out the invention. Thepreferred embodiments of the invention may include these variations, thepresent inventors intended to those specifically described hereinvarious embodiments of the present invention, the same ordinary skill inthe art are well aware of these changes and the expected variation canskillfully use. Can allow the legal scope of the present inventionincludes all modifications and equivalents of the appended claims, thesubject matter referenced, unless otherwise indicated herein or clearlycontradicted by context.

Sequences of PRCP, PREP, and primers used in Examples.

<210> 1  <211> 496  <212> PRT  <213> NP_005031 PRCP  <400> 1 Met Gly Arg Arg Ala Leu Leu Leu Leu Leu Leu Ser Phe Leu Ala Pro 1        5            10          15 Trp Ala Thr Ile Ala Leu Arg Pro Ala Leu Arg Ala Leu Gly Ser Leu        20            25           30 His Leu Pro Thr Asn Pro Thr Ser Leu Pro Ala Val Ala Lys Asn Tyr      35          40            45 Ser Val Leu Tyr Phe Gln Gln Lys Val Asp His Phe Gly Phe Asn Thr   50            55          60 Val Lys Thr Phe Asn Gln Arg Tyr Leu Val Ala Asp Lys Tyr Trp Lys 65           70           75           80 Lys Asn Gly Gly Ser Ile Leu Phe Tyr Thr Gly Asn Glu Gly Asp Ile          85            90           95 Ile Trp Phe Cys Asn Asn Thr Gly Phe Met Trp Asp Val Ala Glu Glu         100          105          110 Leu Lys Ala Met Leu Val Phe Ala Glu His Arg Tyr Tyr Gly Glu Ser     115          120           125 Leu Pro Phe Gly Asp Asn Ser Phe Lys Asp Ser Arg His Leu Asn Phe   130           135         140 Leu Thr Ser Glu Gln Ala Leu Ala Asp Phe Ala Glu Leu Ile Lys His 145          150          155           160 Leu Lys Arg Thr Ile Pro Gly Ala Glu Asn Gln Pro Val Ile Ala Ile          165           170           175 Gly Gly Ser Tyr Gly Gly Met Leu Ala Ala Trp Phe Arg Met Lys Tyr        180          185          190 Pro His Met Val Val Gly Ala Leu Ala Ala Ser Ala Pro Ile Trp Gln     195           200           205 Phe Glu Asp Leu Val Pro Cys Gly Val Phe Met Lys Ile Val Thr Thr   210          215          220 Asp Phe Arg Lys Ser Gly Pro His Cys Ser Glu Ser Ile His Arg Ser 225          230          235           240 Trp Asp Ala Ile Asn Arg Leu Ser Asn Thr Gly Ser Gly Leu Gln Trp          245           250          255 Leu Thr Gly Ala Leu His Leu Cys Ser Pro Leu Thr Ser Gln Asp Ile       260           265          270 Gln His Leu Lys Asp Trp Ile Ser Glu Thr Trp Val Asn Leu Ala Met     275           280           285 Val Asp Tyr Pro Tyr Ala Ser Asn Phe Leu Gln Pro Leu Pro Ala Trp   290           295           300 Pro Ile Lys Val Val Cys Gln Tyr Leu Lys Asn Pro Asn Val Ser Asp 305           310           315          320 Ser Leu Leu Leu Gln Asn Ile Phe Gln Ala Leu Asn Val Tyr Tyr Asn          325          330           335 Tyr Ser Gly Gln Val Lys Cys Leu Asn Ile Ser Glu Thr Ala Thr Ser         340          345          350 Ser Leu Gly Thr Leu Gly Trp Ser Tyr Gln Ala Cys Thr Glu Val Val     355           360           365 Met Pro Phe Cys Thr Asn Gly Val Asp Asp Met Phe Glu Pro His Ser   370         375           380 Trp Asn Leu Lys Glu Leu Ser Asp Asp Cys Phe Gln Gln Trp Gly Val 385          390          395         400 Arg Pro Arg Pro Ser Trp Ile Thr Thr Met Tyr Gly Gly Lys Asn Ile          405           410           415 Ser Ser His Thr Asn Ile Val Phe Ser Asn Gly Glu Leu Asp Pro Trp        420           425            430 Ser Gly Gly Gly Val Thr Lys Asp Ile Thr Asp Thr Leu Val Ala Val      435          440           445 Thr Ile Ser Glu Gly Ala His His Leu Asp Leu Arg Thr Lys Asn Ala   450            455          460 Leu Asp Pro Met Ser Val Leu Leu Ala Arg Ser Leu Glu Val Arg His 465          470          475           480 Met Lys Asn Trp Ile Arg Asp Phe Tyr Asp Ser Ala Gly Lys Gln His          485            490         495  <210> 2  <211> 710  <212> PRT <213> NP_002717 PREP  <400> 2 Met Leu Ser Leu Gln Tyr Pro Asp Val Tyr Arg Asp Glu Thr Ala Val 1        5            10          15 Gln Asp Tyr His Gly His Lys Ile Cys Asp Pro Tyr Ala Trp Leu Glu       20           25            30 Asp Pro Asp Ser Glu Gln Thr Lys Ala Phe Val Glu Ala Gln Asn Lys     35           40           45 Ile Thr Val Pro Phe Leu Glu Gln Cys Pro Ile Arg Gly Leu Tyr Lys    50           55           60 Glu Arg Met Thr Glu Leu Tyr Asp Tyr Pro Lys Tyr Ser Cys His Phe 65          70           75           80 Lys Lys Gly Lys Arg Tyr Phe Tyr Phe Tyr Asn Thr Gly Leu Gln Asn          85           90           95 Gln Arg Val Leu Tyr Val Gln Asp Ser Leu Glu Gly Glu Ala Arg Val       100           105           110 Phe Leu Asp Pro Asn Ile Leu Ser Asp Asp Gly Thr Val Ala Leu Arg     115           120           125 Gly Tyr Ala Phe Ser Glu Asp Gly Glu Tyr Phe Ala Tyr Gly Leu Ser   130           135          140 Ala Ser Gly Ser Asp Trp Val Thr Ile Lys Phe Met Lys Val Asp Gly 145           150          155            160 Ala Lys Glu Leu Pro Asp Val Leu Glu Arg Val Lys Phe Ser Cys Met          165          170           175 Ala Trp Thr His Asp Gly Lys Gly Met Phe Tyr Asn Ser Tyr Pro Gln        180          185          190 Gln Asp Gly Lys Ser Asp Gly Thr Glu Thr Ser Thr Asn Leu His Gln     195           200          205 Lys Leu Tyr Tyr His Val Leu Gly Thr Asp Gln Ser Glu Asp Ile Leu   210           215          220 Cys Ala Glu Phe Pro Asp Glu Pro Lys Trp Met Gly Gly Ala Glu Leu 225          230          235           240 Ser Asp Asp Gly Arg Tyr Val Leu Leu Ser Ile Arg Glu Gly Cys Asp          245          250           255 Pro Val Asn Arg Leu Trp Tyr Cys Asp Leu Gln Gln Glu Ser Ser Gly        260          265           270 Ile Ala Gly Ile Leu Lys Trp Val Lys Leu Ile Asp Asn Phe Glu Gly      275            280           285 Glu Tyr Asp Tyr Val Thr Asn Glu Gly Thr Val Phe Thr Phe Lys Thr   290           295          300 Asn Arg Gln Ser Pro Asn Tyr Arg Val Ile Asn Ile Asp Phe Arg Asp 305          310          315            320 Pro Glu Glu Ser Lys Trp Lys Val Leu Val Pro Glu His Glu Lys Asp          325           330           335 Val Leu Glu Trp Ile Ala Cys Val Arg Ser Asn Phe Leu Val Leu Cys        340           345           350 Tyr Leu His Asp Val Lys Asn Ile Leu Gln Leu His Asp Leu Thr Thr     355           360           365 Gly Ala Leu Leu Lys Thr Phe Pro Leu Asp Val Gly Ser Ile Val Gly   370          375          380 Tyr Ser Gly Gln Lys Lys Asp Thr Glu Ile Phe Tyr Gln Phe Thr Ser 385           390         395            400 Phe Leu Ser Pro Gly Ile Ile Tyr His Cys Asp Leu Thr Lys Glu Glu          405            410           415 Leu Glu Pro Arg Val Phe Arg Glu Val Thr Val Lys Gly Ile Asp Ala       420            425           430 Ser Asp Tyr Gln Thr Val Gln Ile Phe Tyr Pro Ser Lys Asp Gly Thr      435          440            445 Lys Ile Pro Met Phe Ile Val His Lys Lys Gly Ile Lys Leu Asp Gly   450           455            460 Ser His Pro Ala Phe Leu Tyr Gly Tyr Gly Gly Phe Asn Ile Ser Ile 465           470          475          480 Thr Pro Asn Tyr Ser Val Ser Arg Leu Ile Phe Val Arg His Met Gly          485           490           495 Gly Ile Leu Ala Val Ala Asn Ile Arg Gly Gly Gly Glu Tyr Gly Glu         500          505           510 Thr Trp His Lys Gly Gly Ile Leu Ala Asn Lys Gln Asn Cys Phe Asp     515           520           525 Asp Phe Gln Cys Ala Ala Glu Tyr Leu Ile Lys Glu Gly Tyr Thr Ser   530         535           540 Pro Lys Arg Leu Thr Ile Asn Gly Gly Ser Asn Gly Gly Leu Leu Val 545          550           555           560 Ala Ala Cys Ala Asn Gln Arg Pro Asp Leu Phe Gly Cys Val Ile Ala          565          570           575 Gln Val Gly Val Met Asp Met Leu Lys Phe His Lys Tyr Thr Ile Gly        580          585          590 His Ala Trp Thr Thr Asp Tyr Gly Cys Ser Asp Ser Lys Gln His Phe      595          600           605 Glu Trp Leu Val Lys Tyr Ser Pro Leu His Asn Val Lys Leu Pro Glu   610           615          620 Ala Asp Asp Ile Gln Tyr Pro Ser Met Leu Leu Leu Thr Ala Asp His 625          630           635          640Asp Asp Arg Val Val Pro Leu His Ser Leu Lys Phe Ile Ala Thr Leu         645            650          655Gln Tyr Ile Val Gly Arg Ser Arg Lys Gln Ser Asn Pro Leu Leu Ile        660           665           670His Val Asp Thr Lys Ala Gly His Gly Ala Gly Lys Pro Thr Ala Lys     675           680           685Val Ile Glu Glu Val Ser Asp Met Phe Ala Phe Ile Ala Arg Cys Leu   690           695           700 Asn Val Asp Trp Ile Pro 705          710 <210> 3 <211> 23 <212> DNA <213> PRCP forward primer<400> 3 tctacactgg taatgaaggg gac 23 <210> 4 <211> 23 <212> DNA <213>PRCP reverse primer <400> 4 tccttgaatg agttgtcacc aaa 23 <210> 5 <211>21 <212> DNA <213> PREP forward primer <400> 5 gagaccgccg tacaggatta t21 <210> 6 <211> 23 <212> DNA <213> PREP reverse primer <400> 6tgaagtggca actatacttg gga 23

1. A method for treating a disease associated with the PI3K/AKT/mTORsignaling pathway, said method comprising administering to a patient inneed of the treatment an effective dose of a compound selected from thegroup consisting of: a PRCP antagonist, a PREP antagonist, and a PRCPand PREP dual antagonist.
 2. The method of claim 1, wherein the methodfurther comprises administering to the patient an effective amount of anmTOR antagonist.
 3. The method of claim 1, wherein the method comprisesa step of degrading insulin receptor substrate proteins.
 4. (canceled)5. The method of claim 1, wherein the compound is the PRCP and PREP dualantagonist and comprises nucleotides.
 6. The method of claim 1, whereinthe compound is the PRCP and PREP dual antagonist and is selected fromthe group consisting of a tert-butyl (2S)-2-{[(2S)-2-methyl acylpyrroleembankment 1-yl]carbonyl}pyrrolidine-1-carboxylic acid ester andderivatives thereof.
 7. The method of claim 1, wherein the compound isthe PRCP and PREP dual antagonist and is selected from the groupconsisting of [8-(dimethylamino)octylthio]-6-propan-2-yl-3-yl]-thiophene-2-yl methyl ketone and derivativesthereof.
 8. The method of claim 2, wherein the mTOR antagonist comprisesmTOR inhibitory nucleotides.
 9. The method of claim 2, wherein the mTORantagonist is selected from the group consisting of rapamycin andderivatives thereof.
 10. The method of claim 1, wherein the disease iscancer.
 11. The method of claim 1, wherein the disease is aneurodegenerative disease.
 12. The method of claim 1, wherein saidsignal PI3K/AKT/mTOR signaling related disease is a metabolic disease.13. The method of claim 1, wherein the disease is hamartoma syndrome.14. The method of claim 1, wherein the disease is hereditary myopathy.